BIO240 Module 6B Cultivating Microorganisms

Direct Count

Standard light microscopy to count cells directly

Viable cell counts

Counts colonies that form with the pour plate or spread plate methods

• counts colony-forming units (CFUs)

Turbidity Measurements

Uses a spectrophotometer to measure the absorbance of a culture at various time points and a growth curve can be constructed

Growth Curve Components

The stages of microbial growth observed in a culture, typically including lag, log, stationary, and death phases.

Lag phase

The initial period of growth when cells adapt to new conditions before cell division begins.

Exponential phase

growth of a microorganism reaches its maximal rate, generation time is minimized and cells divide at a constant rate, leading to rapid population increase.

Generation time

Amount of time it takes to divide

Stationary phase

After resources run out or waste material builds up to a certain level, microbial growth stops

• death and cell division are balanced

Death phase

Most cells are dying rather than dividing as resources are depleted

Continuous Culture

A method of growing microorganisms where nutrients are continually supplied and waste products are continuously removed, allowing for constant cell population and growth.

Chemostat

System that brings in fresh medium and removes old medium/microbes

Polymer membrane filters

made from cellulose acetate or nitrate with uniform pore diameters

Nucleopore membrane filters

Composed of polycarbonate and very thin. To get nucleopore filters, polycarbonate films are exposed to radiation causing cracks and then enlarged by a chemical etching process

Pre-filter

Removes large particles prior to other forms of filtration

Autoclaves

Uses pressure and heath to sterilize

• pressure itself does not kill

Moist heat permeates a cell very rapidly

goal of pasteurization

reduces microbial load

Pasteurization processes

High temperatures for short times (HTST) → 72C for 15s

Ultra High Temperature → 135C for <1s

Extended Self Life → filter out material and low-temp pasteurization

Effects of freezing on microbes

• Bacteria cannot replicate at low temperatures

• Biochemical reactions do not work efficiently

• Ice crystals form in the foods and break open the microbial cells

Electromagnetic radiation

Eliminate microorganisms from surfaces or for use on materials the allow light to penetrate

UV light impact on DNA

Forms thymine dimers (two T bases occur next to each other and become covalently linked by UV light)

Ionizing Radiation

Sterilizes material by penetrating cells and disrupting molecular structures, including DNA.

Cobalt-60 or Cesium

Nonselective Agents

Toxic to any cells → attacks specific groups on proteins and amino acids within cell

Selective antimicrobial agents

Targeted at specific microbial groups → antibiotics

Bacteriostatic Agents

Do not kill cells but inhibit their growth

Bactericidal Agents

Result in cell death → simply not viable (do not lyse)

Bacteriolytic Agents

Result in cell lysis

Broad-spectrum

Tetracycline → affects gram-negative, gram-positive and intracellular parasitic bacteria

Narrow Spectrum

Polymyxin B, penicillin → Smaller Range

What does penicillin do?

Inhibition of cell wall synthesis

What does tetracycline do

Inhibition of protein synthesis

What does polymyxin B do

Disruption of cell outer membrane

Decimal Reduction Time (D Value)

Time required to kill 90% of the target organism under specific conditions

Decimal Reduction Dose (D10)

Radiation dose required to kill 90% of target organism under specific conditions